Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

• the invention relates to a method for detecting a target biological material contained in a sample, using a capture phase comprising an organic molecule having at least one reactive function, and at least one protein material capable of recognizing or binding. , specifically and directly or indirectly, to the target biological material, said protein material having a specific site of covalent binding to the reactive function of the organic molecule.
• Document EP-A-0 319 012 discloses a method for immobilizing a protein on a solid support, via reactive functions, with the aim of stimulating the proliferation or development of T cells and the production of killer lymphocytes.
• the protein has, on its C-terminal end, a covalent binding site to bind to said reactive functions, which consists of a series of amino acids in which are distributed two to four lysine residues.
• the problem posed by the covalent binding site of this prior art resides in the fact that, in a diagnostic application, the results of detection of a target biological material with which it is capable of binding, are similar to those obtained with protein material not provided with such a binding site.
• a method of demonstrating a target biological material is provided by using at least one capture phase which comprises a protein material having a covalent binding site which allows efficient orientation of said protein material, and leads to a sensitive and efficient detection of said biological material.
• a capture phase which comprises an organic molecule having at least one reactive function and at least one material protein capable of recognizing or binding, specifically and directly or indirectly, to the target biological material, said protein material having a specific site of covalent binding to the reactive function of the organic molecule, which consists of at least one tag comprising at least six lysine residues, or lysine derivative, contiguous, said target biological material is brought into contact with at least the capture phase, and the target biological material fixed on the capture phase is detected.
• target biological material By highlighting a target biological material according to the invention, it is understood the fixing, separation, isolation, detection and / or quantification of this material, the enrichment of a fraction in target biological material, according to a specific or non-specific fixing method, qualitatively and / or quantitatively.
• the capture phase can also comprise a marker, and in this case in particular, it can consist of a detection phase.
• a detection phase which comprises an organic molecule having at least one reactive function, at least one protein material capable of recognizing or of binding, specifically and directly or indirectly, to the material target biological, and a marker, said protein material having a specific binding site by covalent to the reactive function of the organic molecule, which consists of at least one tag comprising at least six lysine residues, or lysine derivative, contiguous.
• a sample as understood according to the invention includes any sample likely to contain biological material, in particular a sample such as that obtained from a biological fluid, a sample of food origin, or a cell culture. .
• the sample consists of all or part of another sample, in particular it may consist of an aliquot, a dilution.
• a protein material according to the invention comprises proteins, in particular recombinant proteins, in particular antigens, antibodies, peptides such as synthetic peptides.
• the method of the invention could also be implemented with equipment such as the nucleic acid peptide analogs (PNA).
• PNA nucleic acid peptide analogs
• organic molecule is meant a molecule of variable size; thus we understand both a small molecule such as a hapten and a macromolecule such as a polymer.
• the method of the invention may comprise, before the step of detecting the target biological material, a step of fixing the organic molecule on a carrier molecule.
• the hapten is biotin and the carrier molecule is avidin.
• a polymer as used according to the invention is a polymer in particulate or linear form.
• It may be a homopolymer especially chosen from polylysine and polytyrosine, or else a copolymer especially chosen from maleic anhydride copolymers, N-vinyl-pyrrolidone copolymers, polysaccharides, natural or synthetic, polynucleotides and copolymers of amino acids such as enzymes.
• it is a copolymer chosen from maleic anhydride / methyl-vinyl-ether copolymer, N-vinyl-pyrrolidone / N-acryloxysuccinimide copolymer, poly-6-aminoglucose, and enzymes such as horseradish peroxidase (HRP). or alkaline phosphatase or their derivatives carrying at least one reactive function.
• HRP horseradish peroxidase
• reactive function of the organic molecule is understood to mean either a reactive function chosen in particular from the ester, halocarbonyl, sulfhydrile, disulfide, epoxide, haloalkyl and aldehyde functions; or a function activatable by an activating agent such as carbodiimides or homo- or hetero-bifunctional compounds.
• an activatable function is in particular chosen from the acid, amino and hydroxyl functions.
• a tag can be defined as a reported amino acid sequence, that is to say added to the original structure of the protein material, which is introduced in a privileged place of said original structure to allow it to be exposed in a way relevant, in particular with regard to its covalent attachment to the organic molecule.
• a covalent binding site of the protein material under consideration may consist of a tag comprising six or more lysine residues, or lysine derivative, and possibly other amino acids, or may consist of several of said tags .
• lysine derivative it is understood that lysine can be chemically modified, provided that these modifications substantially preserve or even develop the specificity of the covalent binding site.
• the tag (s) described above can be found anywhere in the primary structure of the protein material. Preferably, it is located on the N- or C-terminal end of the protein material.
• the protein material comprises an antigen which specifically recognizes said antigen; - if the target biological material is an antigen, the protein material comprises an antibody which specifically recognizes said antigen.
• the invention relates to a phase for capturing a target biological material which comprises an organic molecule having at least one reactive function and at least one protein material capable of recognizing or of specifically and directly or indirectly binding to the target biological material, said protein material having a specific site for covalently binding to the reactive function of the organic molecule, which consists of at least one tag comprising at least six contiguous lysine residues; preferably, the organic molecule is a polymer or a hapten.
• An advantageous polymer is particulate or linear and in particular chosen from homopolymers such as polylysine, polytyrosine; and copolymers such as maleic anhydride copolymers, N-vinyl-pyrrolidone copolymers, polysaccharides, natural or synthetic, polynucleotides and copolymers of amino acids such as enzymes. More advantageously still, the polymer is chosen from maleic anhydride / methyl-vinyl-ether copolymer, N-vinyl-pyrrolidone / N-acryloxysuccinimide copolymer, poly-6-aminoglucose, horseradish peroxidase (HRP) and alkaline phosphatase .
• homopolymers such as polylysine, polytyrosine
• copolymers such as maleic anhydride copolymers, N-vinyl-pyrrolidone copolymers, polysaccharides, natural or synthetic, polynucleo
• the capture phase can also comprise an appropriate carrier molecule.
• the hapten is biotin, and where appropriate the carrier molecule is avidin.
• the tag as defined above is placed on the N- or C-terminal end of the protein material.
• the reactive function of the organic molecule is in particular chosen from the ester, acid, halocarbonyl, sulfhydrile, disulfide, epoxide, haloalkyl and aldehyde functions.
• the invention also relates to a phase for detecting a target biological material, having the same characteristics as the capture phase above and further comprising a marker.
• the organic molecule can be a polymer or a hapten.
• the organic molecule consisting of hapten also represents the marker.
• the marker for the detection phase is preferably chosen from the group consisting of an enzyme, a protein, a peptide, an antibody, a hapten such as biotin, 1 * iminobiotin, a fluorescent compound such as rhodamine, a radioactive compound , a chemiluminescent compound, an electrodensity component, a magnetic component and the like.
• Another object of the invention is a reagent for the detection of a target biological material, comprising a capture phase of the invention, and / or a detection phase of the invention.
• the capture phase is fixed, directly or indirectly, on a solid support, by passive adsorption or by covalence.
• the solid support can be in any suitable form such as a plate, a cone, an optionally radioactive and / or fluorescent and / or magnetic and / or conductive ball, a strip, a glass tube, a well, a sheet, a chip or the like. It is chosen from polystyrenes, styrene-butadiene copolymers, styrene-butadiene copolymers in admixture with polystyrenes, polypropylenes, polycarbonates, polystyrene-acrylonitrile copolymers, styrene-methylmethacrylate copolymers, among synthetic and natural fibers, among polysaccharides and cellulose derivatives, glass, silicon and their derivatives.
• the capture phase comprises an organic molecule which comprises or consists of an anti-tag antibody and at least one protein material capable of recognizing or binding , specifically and directly or indirectly, to the target biological material, said protein material having a specific site for binding to the organic molecule, which consists of at least one tag comprising at least six lysine residues, or lysine derivative, contiguous.
• Such a method may further comprise the use of a detection phase, having the characteristics of the capture phase as it has just been defined, and further comprising a marker.
• This marker can advantageously consist of said antibody.
• FIG. 2 represents a comparison graph between the stability as a function of time (in days), of the conjugate polymer (AMVE) / protein (RH24K) (curve>) and that of the free RH24K protein (curve •).
• Figure 3 shows a diagram summarizing the "sandwich" detection technique.
• FIG. 4 represents a graph translating the immunoreactivity of the polymer / RH24 (curve •) and polymer / RH24K (curve •) conjugate compounds, vis-à-vis a monoclonal antibody.
• FIG. 5 represents a graph translating the immunoreactivity of the polymer / RH24 (curve •) and polymer / RH24K (curve B) conjugates, vis-à-vis a serum.
• Figure 6 illustrates the results of a sandwich and indirect ELISA test with the IG2D4 antibody, using a graph representing the OD as a function of the antibody titer.
• Figure 7 illustrates the results of an indirect ELISA test with the IG2D4 antibody, using a graph representing the OD as a function of the antibody titer.
• PMH24 (Cheynet et al. 1993) was modified by insertion of a synthetic adapter 3 'to the p24 gene in order to express RH24K, corresponding to RH24 which has a specific site on its C-terminal end of covalent bonding called "polylysine tail" and constituted by 6 contiguous lysine residues.
• the adapter was previously produced by hybridization of two oligonucleotides having the following sequences respectively: SEQ ID NO: 1:
• the protein is expressed under the control of the Tac promoter in the bacterial strain E. coli XLI (see above).
• a preculture of E. coli XLI, containing the expression vector, is previously carried out overnight at 37 ° C. in Luria broth (LB) in the presence of ampicillin (amp; 50 ⁇ g / ml) and tetracycline (tet; 12 ⁇ g / ml ) in order to seed LB-amp-tet medium (1/40 to 1/25); the latter is cultured until an OD at 600 nm of 0.6.
• the expression of the protein is then induced by the addition of isopropyl- / 3-D-thiogalactopyranoside (IPTG) to the concentration of 1 mM in the medium. for 2 h 30 at 37 ° C with stirring. After centrifugation at 5000 revolutions / min (rotor JA21 Beckman) for 10 minutes at 4 ° C, the bacteria are taken up in a Tris-HCl 50 mM EDTA buffer, 1 mM NaCl 100 mM MgCl2 10 mM pH 8 (3 mL / g of biomass) in the presence of protease inhibitor (aprotinin 2 ⁇ g / ml and leupeptin 2 ⁇ g / ml) then lysed by sonication at 0 ° C.
• IPTG isopropyl- / 3-D-thiogalactopyranoside
• the soluble fraction is taken up by centrifugation at 10,000 rpm (rotor JA21, Beckman) for 15 minutes at 4 ° C and then deposited on an affinity column by metal chelation for the purification of RH24K (Chelating Sepharose Fast Flow no. 17057501).
• the gel was previously loaded with Zn 2+ according to the supplier's recommendations and then equilibrated in 67 mM sodium phosphate buffer NaCl pH 7, 8 for the purpose of purification. After depositing the supernatant, the column is successively washed with 67 mM sodium phosphate buffer NaCl pH 7.8 and then 200 mM ammonium acetate 0.5 M NaCl pH 6.
• the RH24K is eluted with 200 ammonium acetate mM 0.5 M NaCl pH 4.
• the fractions collected are dialyzed against 50 mM sodium phosphate buffer pH 7.8.
• the purified protein is characterized by electrophoresis on SDS-PAGE and by passage through gel filtration / HPLC. Its purity is greater than 90% therefore comparable to the purity of RH24. Analyzed in mass spectrometry, its molecular weight is 28027 compatible with the theoretical molecular weight 27992. The protein is recognized by anti-p24 polyclonal and monoclonal antibodies in Western Blot and in ELISA.
• reaction medium is stirred for 3 hours at 37 ° C.
• the progress of the reaction is monitored by HPLC, on a Waters steric exclusion column with a 0.1 M phosphate buffer, pH 6.8, as mobile phase.
• AMVE 65 poly (methylvinyl er / maleic anhydride)
• PEAM 86 poly (ethylene / maleic anhydride)
• SAM 49 poly (styrene / maleic anhydride)
• NVPAM 36 poly (-vinylpyrrolidone / maleic anhydride)
• the conjugate compounds produced are biotinylated p24 (RH24 or RH24K) which will be used for detection in the p24 antigen sandwich test, in accordance with the diagram shown in FIG. 3.
• Biotin couplings are carried out on RH24K and on RH24, checked and then analyzed in an antigen sandwich test so as to define coupling conditions favorable to RH24K.
• Proteins are used at a concentration of 1 mg / ml.
• Different buffers were tested on the basis of the buffers tested during the p24 / AMVE couplings: borate 0.1 M pH 9.2, Tris 0.1 M pH 7.2, phosphate 0.1 M pH 7.2 and carbonate 0 , 1 M pH 8.5.
• the following biotin / p24 ratios were tested: 5/1, 10/1, 25/1, 50/1, 100/1.
• the couplings were incubated for 1 hour at 37 ° C.
• the conjugated p24 / biotin compounds were used for detection according to the scheme represented in FIG. 3 with monoclonal antibodies and a positive serum.
• the conjugated compounds RH24 / biotin and RH24K / biotin show activity in antigen sandwich test with monoclonal antibodies and positive serum.
• FIGS. 4 and 5 show in the form of a graph, the compared responses of the conjugated compounds obtained with RH24K and RH24 at different levels of functionalization in biotin.
• FIG. 4 relates to the results obtained with a monoclonal antibody, a test carried out on a micro-titration plate.
• the Figure 5 represents the results obtained on serum with the Vidas immunoanylyse automaton from bioMérieux.
• RH24K has a higher signal than RH24 regardless of the experimental conditions tested. There is an optimal biotin / p24 ratio of 25/1 for monoclonal antibodies, and 10/1 for serum.
• Example 6 Influence of the lysine residue composition of the poly-lvsine tag on the coupling efficiency
• the biological material used to evaluate the influence of the lysine residue composition of the poly-lysine tag on the coupling efficiency is as follows:
• the recombinant protein RH24K comprises, in its amino-terminal part, a poly-histidine tag [MRGS (H) gGSVDESM] used for its purification by chelation with a metal ion, and, in its carboxy terminal part, a poly-lysine tag [PG (K ) gSVDESL] dedicated to coupling;
• MRGS (H) gGSVDESM poly-histidine tag
• PG (K ) gSVDESL poly-lysine tag dedicated to coupling
• the recombinant protein RHK24 comprises, in its amino-terminal part, a poly-lysine tag [MRGSCH (K) 2 HH (K) 2 HH (K) 2 GSVDESM]; this protein can be represented as follows:
• the recombinant protein R24 contains neither the carboxy-terminal poly-lysine tag nor the amino-terminal poly-histidine tag; we can represent it by p24.
• the polymers used are AMVE (maleic anhydride-co-methylvinyl ether). The coupling conditions are identical for the three proteins.
• the coupling yield obtained with the RH24K protein (6 contiguous lysine residues) is much higher than the coupling yield obtained with the RHK24 protein (6 lysine residues in three blocks of 2).
• the biological material used to obtain anti-tag antibodies capable of recognizing this tag in the context of a fusion with a recombinant protein is as follows:
• the recombinant protein RH24K comprises, in its amino-terminal part, a poly-histidine tag [MRGS (H) 6 GSVDESM] used for its purification by chelation with a metal ion and in its carboxy terminal part a poly-lysine tag [PG (K ) 6 SVDESL] dedicated to coupling and represented by
• the biological material used to select the antibodies produced is as follows: - the recombinant protein RH24K
• the recombinant protein RH24 contains neither the carboxy-terminal poly-lysine tag, nor the amino-terminal poly-histidine tag, and represented by p24.
• a sandwich ELISA test comprising, in the capture phase, a goat anti-mouse antibody and, in the detection phase, the RH24K or R24 antigens revealed by an anti-24 monoclonal antibody coupled to peroxidase;
• an indirect ELISA test comprising in the capture phase, the RH24K or R24 or P400 antigens and, in the detection phase, a goat anti-mouse IgV antibody coupled to peroxidase.
• mice were immunized according to the following protocol: 3 intraperitoneal injections, with, for the first injection, the RH24K protein, and for the following two injections, the peptide 400 coupled to KLH. Mergers were performed and screenings performed using the sandwich and indirect ELISA tests described above.
• MRGS (H) 6 GSVDESM also comprising the motif SVDESL and the protein p24.
• FIGS. 6 and 7 illustrate the results of the sandwich and indirect ELISA tests for the IG2D4 antibody.
• FIG. 6 illustrates a sandwich ELISA test and represents the OD as a function of the titer (dilution) of the ascites of the IG2D4 antibody ; the RH24K and R24 recombinant proteins, constituting the detection phase, are used at a concentration of 0.1 ⁇ g / ml.
• FIG. 6 illustrates a sandwich ELISA test and represents the OD as a function of the titer (dilution) of the ascites of the IG2D4 antibody ; the RH24K and R24 recombinant proteins, constituting the detection phase, are used at a concentration of 0.1 ⁇ g / ml.
• FIG. 6 illustrates a sandwich ELISA test and represents the OD as a function of the titer (dilution) of the ascites of the IG2D4 antibody ; the R
• FIG. 7 illustrates an indirect ELISA test and represents the OD as a function of the titer (dilution) of the ascites of the antibody IG2D4; the recombinant proteins RH24K and R24, constituting the capture phase, are used at the concentration of 0.5 ⁇ g / ml and the synthetic peptide P400 at the concentration of 0.05 ⁇ g / ml.

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Citations (4)

• 1997
  • 1997-06-20 FR FR9708055A patent/FR2764988B1/fr not_active Expired - Fee Related
• 1998
  • 1998-06-19 EP EP98932247A patent/EP0920626B1/de not_active Expired - Lifetime
  • 1998-06-19 ES ES98932247T patent/ES2191316T3/es not_active Expired - Lifetime
  • 1998-06-19 AT AT98932247T patent/ATE233405T1/de not_active IP Right Cessation
  • 1998-06-19 JP JP50389999A patent/JP3880633B2/ja not_active Expired - Lifetime
  • 1998-06-19 CA CA002263756A patent/CA2263756A1/fr not_active Abandoned
  • 1998-06-19 WO PCT/FR1998/001299 patent/WO1998059241A1/fr active IP Right Grant
  • 1998-06-19 AU AU82209/98A patent/AU748402B2/en not_active Ceased
  • 1998-06-19 DE DE69811614T patent/DE69811614T2/de not_active Expired - Lifetime
  • 1998-06-19 US US09/230,846 patent/US6632615B2/en not_active Expired - Lifetime
  • 1998-06-19 BR BR9806053-8A patent/BR9806053A/pt not_active IP Right Cessation

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